Novel luminescent dyes for confocal laser scanning microscopy used in parasite Trematoda diagnostics*

Abstract

Benzanthrone derivates are now widely used in many industrial and scientific applications as dyes for polymers and textiles. In biochemical, biomedical and diagnostics investigations benzanthrone dyes are used as a lipophilic fluorescent probe since many benzanthrone derivates demonstrate bright fluorescence and they have ability to intercalate between membrane lipids. The aim of research presented here was to assess the luminescence ability of benzanthrone derivatives using microscopic visualization of biological objects. Accordingly, specimens of freshwater trematodes: Diplostomum spathaceum, Diplodiscus subclavatus and Prosotocus confusus, were stained by novel benzanthrone dyes using different fixatives. The samples were examined under a confocal laser scanning microscope. All of the dyes tested demonstrated good results for digestive and reproductive system visualization. Based on obtained results we conclude that benzanthrone dyes could be used for internal and external structure confocal laser scanning microscopic imaging of trematode specimens.

A Simple and Rapid Staining Technique for Sex Determination of Trichinella Larvae Parasites by Confocal Laser Scanning Microscopy

“…Currently, the synthesis of new high-emitting and low-toxic luminescent dyes (in particular, benzanthrone derivatives) remains an important task for the search of new efficient probes and labels. Recently reported derivatives of 3-acetamidobenzanthrone are highly luminescent compounds and used as sensors for various substances (Wang et al, 2004; Staneva & Betcheva, 2007; Staneva et al, 2019), examination of adult and larvae of trematodes from aquatic animals (Kirilova et al, 2018 a ), and visualization of callus embryo (Kirilova et al, 2019).…”

“…The aim of the present study was to develop a staining technique using the synthesized benzanthrone luminescent dye for microscopic examination and sex determination of T. britovi and T. spiralis larvae by CLSM. In previous studies, by Kirilova et al (2018 a ), fluorescent benzanthrone dyes 3- N -( N ′, N ′-dimethylbenzamidino)benzanthrone, 3- N -( N ′, N ′-dimethylacetamidino)benzanthrone, 3- N -[ N ′-(4-methylphenyl)acetamidino]benzanthrone, and (3- N -piperidinobenzanthrone) with fixatives AFA and ethanol were applied for the visualization of surface morphological features and organ systems of various species of adult and larvae of trematodes from aquatic animals and showed good staining properties for digestive and reproductive system imaging. In the present study, the synthesized dye with an amide group which is more suitable for the examination of Trichinella larvae was found.…”

Comparison of the Benzanthrone Luminophores: They Are Not Equal for Rapid Examination of Parafasciolopsis fasciolaemorpha (Trematoda: Digenea)

“…In literature, there are described various Trematoda stainings methods such as Gower's carmine [24], Ehrlich's acid hematoxylin and Celestin blue-b [13] actin-antibodies and fluorescently labeled phalloidin staining [46][47][48]. Recently we reported that benzanthrone dyes are a useful tool for imaging freshwater trematodes [3], for callus embryo determination [4] and for sex determination of Trichinella larva [49]. Therefore, we continue our research on the synthesis of new benzanthrone markers that bind to actin elements to determine internal organs and systems of trematodes.…”

“…These substances luminescence intensely, have high quantum yields, and their spectral properties are sensitive to the local microenvironment's polarity enabling sensing applications. The spectral properties of the developed fluorescent dyes were investigated in detail [3,4]. As a result, two promising compounds were selected for the current research of the possibilities of studying parasites.…”

“…Therefore, improvements in dye chemistry are required for the discovery of the helminth's detailed structure. In a previous investigation by Kirilova et al [3], various benzanthrone derivatives (with substituted amidine or amine groups in 3-position of benzanthrone core) were applied for visualization of the internal and external structure of freshwater trematodes species such as Diplostomum spathaceum, Diplodiscus subclavatus and Prosotocus confusus. Studied benzanthrone dyes, using AFA fixative, showed good visualization of internal organ systems and body wall of parasites.…”